With the aid of specific monoclonal antibodies, an immunohistochemical technique has recently been developed for the detection of intratumoral thymidylate synthase (TS). This technique can be applied to paraffin-embedded material suitable for retrospective studies. In order to examine this technique further, the TS enzyme activity of lysates from frozen-stored colorectal cancer (CRC) specimens were compared with their immunohistochemical TS staining intensity (arbitrarily graded from 0 to 3). A statistically significant correlation between these two methods on a total of 25 tumour specimens (P < 0.001) was observed. The staining intensity in different areas of 48 paraffin-embedded CRCs was examined. Sixty-seven per cent of the tumours were homogeneously stained (either grades 0-1 or 2-3), 33% showed a heterogeneity in TS staining. Increased TS expression correlated with more advanced Dukes' stage (P < 0.001). It is concluded that TS immunostaining intensity reflects TS enzyme activity in colorectal tumours and is well suited for paraffin-embedded material. The TS immunostaining pattern is heterogeneous in up to one-third of the tumours.